Search results for "Fluorescence"
showing 10 items of 2463 documents
Multi-approach metabolomics analysis and artificial simplified phytocomplexes reveal cultivar-dependent synergy between polyphenols and ascorbic acid…
2017
Fruits of the sweet cherry (Prunus avium L.) accumulate a range of antioxidants that can help to prevent cardiovascular disease, inflammation and cancer. We tested the in vitro antioxidant activity of 18 sweet cherry cultivars collected from 12 farms in the protected geographical indication region of Marostica (Vicenza, Italy) during two growing seasons. Multiple targeted and untargeted metabolomics approaches (NMR, LC-MS, HPLC-DAD, HPLC-UV) as well as artificial simplified phytocomplexes representing the cultivars Sandra Tardiva, Sandra and Grace Star were then used to determine whether the total antioxidant activity reflected the additive effects of each compound or resulted from synergis…
Fluorogenic Tetrazine-Siliconrhodamine Probe for the Labeling of Noncanonical Amino Acid Tagged Proteins
2018
Tetrazine-bearing fluorescent labels enable site-specific tagging of proteins that are genetically manipulated with dienophile modified noncanonical amino acids. The inverse electron demand Diels-Alder reaction between the tetrazine and the dienophile fulfills the criteria of bioorthogonality allowing fluorescent labeling schemes of live cells. Here, we describe the detailed synthetic and labeling protocols of a near infrared emitting siliconrhodamine-tetrazine probe suitable for super-resolution imaging of residue-specifically engineered proteins in mammalian cells.
Modulation of mitochondriotropic properties of cyanine dyes by in organello copper-free click reaction
2017
Cyanine (Cy) dyes show a general propensity to localize in polarized mitochondria. This mitochondriotropism was used to perform a copper-free click reaction in the mitochondria of living cells. The in organello reaction of dyes Cy3 and Cy5 led to a product that was easily traceable by Forster resonance energy transfer (FRET). As determined by confocal laser scanning microscopy, the Cy3-Cy5 conjugate showed enhanced retention in mitochondria, relative to that of the starting compounds. This enhancement of a favorable property can be achieved by synthesis in organello, but not outside mitochondria.
FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.
2021
International audience; Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever- increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among d…
Hydrogen Bond Fluctuations Control Photochromism in a Reversibly Photo-Switchable Fluorescent Protein
2015
Reversibly switchable fluorescent proteins (RSFPs) are essential for high-resolution microscopy of biological samples, but the reason why these proteins are photochromic is still poorly understood. To address this problem, we performed molecular dynamics simulations of the fast switching Met159Thr mutant of the RSFP Dronpa. Our simulations revealed a ground state structural heterogeneity in the chromophore pocket that consists of three populations with one, two, or three hydrogen bonds to the phenolate moiety of the chromophore. By means of non-adiabatic quantum mechanics/molecular dynamics simulations, we demonstrated that the subpopulation with a single hydrogen bond is responsible for of…
The Unfolded Protein Response Plays a Predominant Homeostatic Role in Response to Mitochondrial Stress in Pancreatic Stellate Cells.
2016
Activated pancreatic stellate cells (PaSC) are key participants in the stroma of pancreatic cancer, secreting extracellular matrix proteins and inflammatory mediators. Tumors are poorly vascularized, creating metabolic stress conditions in cancer and stromal cells that necessitate adaptive homeostatic cellular programs. Activation of autophagy and the endoplasmic reticulum unfolded protein response (UPR) have been described in hepatic stellate cells, but the role of these processes in PaSC responses to metabolic stress is unknown. We reported that the PI3K/mTOR pathway, which AMPK can regulate through multiple inputs, modulates PaSC activation and fibrogenic potential. Here, using primary a…
Flow Cytometric Analysis of the Expression Pattern of Peroxisomal Proteins, Abcd1, Abcd2, and Abcd3 in BV-2 Murine Microglial Cells
2017
Microglial cells play important roles in neurodegenerative diseases including peroxisomal leukodystrophies. The BV-2 murine immortalized cells are widely used in the context of neurodegenerative researches. It is therefore important to establish the expression pattern of peroxisomal proteins by flow cytometry in these cells. So, the expression pattern of various peroxisomal transporters (Abcd1, Abcd2, Abcd3) contributing to peroxisomal β-oxidation was evaluated on BV-2 cells by flow cytometry and complementary methods (fluorescence microscopy, and RT-qPCR). By flow cytometry a strong expression of peroxisomal proteins (Abcd1, Abcd2, Abcd3) was observed. These data were in agreement with tho…
E-beam crosslinked nanogels conjugated with monoclonal antibodies in targeting strategies
2017
Abstract Poly(N-vinyl pyrrolidone)-based-nanogels (NGs), produced by e-beam irradiation, are conjugated with monoclonal antibodies (mAb) for active targeting purposes. The uptake of immuno-functionalized nanogels is tested in an endothelial cell line, ECV304, using confocal and epifluorescence microscopy. Intracellular localization studies reveal a faster uptake of the immuno-nanogel conjugate with respect to the ‘bare’ nanogel. The specific internalization pathway of these immuno-nanogels is clarified by selective endocytosis inhibition experiments, flow cytometry and confocal microscopy. Active targeting ability is also verified by conjugating a monoclonal antibody which recognizes the αv…
A practical algorithmic approach to mature aggressive B cell lymphoma diagnosis in the double/triple hit era. Selecting cases, matching clinical bene…
2019
An accurate diagnosis of clinically distinct subgroups of aggressive mature B cell lymphomas is crucial for the choice of proper treatment. Presently, precise recognition of these disorders relies on the combination of morphological, immunophenotypical, and cytogenetic/molecular features. The diagnostic workup in such situations implies the application of costly and time-consuming analyses, which are not always required, since an intensified treatment option is reasonably reserved to fit patients. The Italian Group of Haematopathology proposes herein a practical algorithm for the diagnosis of aggressive mature B cell lymphomas based on a stepwise approach, aimed to select cases deserving mo…
Identification of metastasis-related genes by genomic and transcriptomic studies in murine melanoma.
2021
Abstract Aims We systematically characterized metastatic murine B16-F10 melanoma, a sub-line derived from murine melanoma B16-F1 cells. Materials and methods RNA-sequencing and network analyses (Ingenuity Pathway Analysis) were performed to identify novel potential metastasis mechanisms. Chromosomal aberrations were identified by multicolor fluorescence in situ hybridization (mFISH) using all 21 murine whole chromosome painting probes. Key findings Numerous genes were overexpressed in B16-F10 cells, some of which have been already described as being metastasis-linked. Nr5a1/sf1, a known prognostic marker for adrenal tumors, was 177-fold upregulated in B16-F10 cells compared to B16-F1 cells.…